The genome of the rice planthopper egg parasitoid wasps Anagrus nilaparvatae casts light on the chemo- and mechanosensation in parasitism.

BACKGROUND: Mymaridae is an ancient insect group and is a basal lineage of the superfamily Chalcidoidea. Species of Mymaridae have great potential for biological control. Anagrus nilaparvatae, a representative species of Mymaridae, is ideal for controlling rice planthopper due to its high rate of parasitism and ability to find hosts efficiently in paddy ridges and fields. RESULTS: Using both PacBio single-molecule real-time and Illumina sequencing, we sequenced and assembled the whole genome of A. nilaparvatae, a first for the family Mymaridae. The assembly consists of 394 scaffolds, totaling 488.8 Mb. The assembly is of high continuity and completeness, indicated by the N50 value of 25.4 Mb and 98.2% mapping rate of Benchmarking Universal Single-Copy Orthologs. In total, 16,894 protein-coding genes in the genome were annotated. A phylogenomic tree constructed for A. nilaparvatae and other 12 species of Hymenoptera confirmed that the family Mymaridae is sister to all remaining chalcidoids. The divergence time between A. nilaparvatae and the other seven Chalcidoidea species was dated at ~ 126.9 Mya. Chemoreceptor and mechanoreceptor genes are important in explaining parasitic behavior. We identified 17 odorant binding proteins, 11 chemosensory proteins, four Niemann-Pick type C2 proteins, 88 olfactory receptors, 12 gustatory receptors, 22 ionotropic receptors and 13 sensory neuron membrane proteins in the genome of A. nilaparvatae, which are associated with the chemosensory functions. Strikingly, there is only one pickpocket receptors and nine transient receptor potential genes in the genome that have a mechanosensory function. CONCLUSIONS: We obtained a high-quality genome assembly for A. nilaparvatae using PacBio single-molecule real-time sequencing, which provides phylogenomic insights for its evolutionary history. The small numbers of chemo- and mechanosensory genes in A. nilaparvatae indicate the species-specific host detection and oviposition behavior of A. nilaparvatae might be regulated by relatively simple molecular pathways.

Genetic Diversity and Population Structure of Gynaephora qinghaiensis in Yushu Prefecture, Qinghai Province Based on the Mitochondrial COI Gene.

Gynaephora qinghaiensis (Lepidoptera: Lymantriidae: Gynaephora), a serious economic pest in alpine meadows, is mainly distributed in Yushu prefecture, Qinghai province, China. In this study, we aimed to investigate the genetic diversity and population structure of G. qinghaiensis through analyzing the sequence of 194 mitochondrial cytochrome oxidase subunit (COI) genes (658 bp in length) identified from 10 geographic populations located in three different countries, including Zhiduo, Zaduo, and Chengduo, of Yushu prefecture. Eleven haplotypes were identified from all populations of G. qinghaiensis with high levels of haplotype diversity (0.78500) and low levels of nucleotide diversity (0.00511). High levels of genetic differentiation and low levels of gene flow were also detected among the populations of G. qinghaiensis. Analysis of molecular variance (AMOVA) showed that 90.13% of the variation was attributed to distribution among groups (Chengduo, Zhiduo, and Zaduo), and 5.22% and 4.65% were, respectively, attributed to distribution among populations, within group, and within populations. The result of mantel test showed a highly significant positive correlation (P < 0.01) between FST and geographical distance. A maximum likelihood tree showed that most haplotypes were grouped into three clusters corresponding to the three counties, suggesting a significant phylogeographic structure in the populations of G. qinghaiensis. The haplotype networks revealed that H2 may be the most primitive haplotype and the most adaptable in nature. Populations 7# and 8# had haplotype H2 and higher haplotype diversity; therefore, we speculated that the G. qinghaiensis in both populations were more adaptable to the environment and had greater outbreak potential and, therefore, should be focused on in terms of prevention and control. Our findings provide valuable information for further study of the population structure and phylogeny of G. qinghaiensis and provide a theoretical basis for the control of G. qinghaiensis.

Prepupae and pupae transcriptomic characterization of Trichogramma chilonis.

The egg parasitoid, Trichogramma chilonis, has significant control effects on agriculture and forestry pests and is widely employed in southern China for the biological control of lepidopteran pests. In this study, transcriptomic analysis was used to gain a clear understanding of the molecular changes in prepupae and pupae of T. chilonis. A total of 16.88 Gb of clean data were obtained and finally assembled into 43,136 unigenes, 18,880 of which were annotated. After FPKM standardization, 117 and 838 specific expression genes were found in prepupae and pupae, respectively. There were 3129 differentially expressed genes between prepupae and pupae. Compared to pupae, 806 genes were up-regulated and 2323 were down-regulated in prepupae. Background on the T. chilonis transcriptome, the enriched GO function and KEGG pathway analysis of DEGs were considered. As indicated by GO classification, up-regulated genes were mainly involved in chitin metabolism, cell adhesion and endocytic, while most down-regulated genes were involved in synthesis of cell components, ion transport and biological regulation. KEGG enrichment analysis showed that 458 DEGs were enriched in 94 metabolic pathways. DEGs involved in nucleotide replication and transcription, substance metabolism, insect hormone biosynthesis, cell growth and death, reproductive metabolism, circadian rhythms and signal transduction pathways were up-regulated or down-regulated to different degrees, indicating that these genes played important roles during the process of metamorphosis in T. chilonis. This study provides a rich data source for the future study of T. chilonis molecular and biological mechanisms. A large number of genes related to metamorphosis were found based on comparison analysis between prepupae and pupae transcriptomes. This study lays a good foundation for in-depth study of gene transcription and regulation mechanisms during T. chilonis metamorphosis.

1H NMR spectroscopy-based metabolic profiling of Ophiocordyceps sinensis and Cordyceps militaris in water-boiled and 50% ethanol-soaked extracts.

Ophiocordyceps sinensis, a well-known Chinese complementary herb, is a rare and valuable therapeutic resource. Cordyceps militaris (C. militaris) is a commonly used substitute for O. sinensis. A metabolomic-based approach for exploring the similarities and differences in the metabolites of O. sinensis and C. militaris in water-boiled and 50% ethanol-soaked extracts is of great significance. To distinguish between the global metabolite profiles of O. sinensis and C. militaris extracts obtained from either the water-boiled or 50% ethanol-soaked methods, we investigated the herb samples using 1HNMR-based metabolic fingerprints combined with multivariate statistical analysis. This study revealed that a total of 52 primary metabolites were identified and quantified from O. sinensis and C. militaris samples. Forty-three (83% of 52) metabolites were detectable in both O. sinensis and C. militaris. According to the variable importance in projection (VIP) value and p-value from the Mann-Whitney test, 7 metabolites (alanine, aspartate, glutamate, mannitol, ornithine, serine, and trehalose) differed between O. sinensis and C. militaris. Arginine, glucose, putrescine, pyroglutamate, betaine, O-phosphocholine, and xylose differed significantly between the water-boiled and 50% ethanol-soaked methods used to prepare the herb extracts. This study demonstrated that water-boiled extraction was a much faster method (30 min. vs 360 days) that resulted in a 30% higher number of extracted metabolites (compared to 50% for the ethanol-soaked method) for both O. sinensis and C. militaris.

Identification and functional characterization of D-fructose receptor in an egg parasitoid, Trichogramma chilonis.

In insects, the gustatory system has a critical function not only in selecting food and feeding behaviours but also in growth and metabolism. Gustatory receptors play an irreplaceable role in insect gustatory signalling. Trichogramma chilonis is an effective biocontrol agent against agricultural insect pests. However, the molecular mechanism of gustation in T. chilonis remains elusive. In this study, we found that T. chilonis adults had a preference for D-fructose and that D-fructose contributed to prolong longevity and improve fecundity. Then, We also isolated the full-length cDNA encoding candidate gustatory receptor (TchiGR43a) based on the transcriptome data of T. chilonis, and observed that the candidate gustatory receptor gene was expressed from the larval to adult stages. The expression levels of TchiGR43a were similar between female and male. A Xenopus oocyte expression system and two-electrode voltage-clamp recording further verified the function analysis of TchiGR43a. Electrophysiological results showed that TchiGR43a was exclusively tuned to D-fructose. By the studies of behaviour, molecular biology and electrophysiology in T. chilonis, our results lay a basic fundation of further study on the molecular mechanisms of gustatory reception and provide theoretical basis for the nutritional requirement of T. chilonis in biocontrol.

Analysis of the Gynaephora qinghaiensis pupae immune transcriptome in response to parasitization by Thektogaster sp.

As a pest on the Qinghai-Tibet Plateau, Gynaephora qinghaiensis causes severe damage to grassland vegetation and its pupae are also natural hosts of Thektogaster sp. To successfully parasitize, endoparasitoids generally introduce or secrete multiple parasitic factors into the host body during the spawning stage to suppress the host immune response. To study the parasitic effects of Thektogaster sp. on G. qinghaiensis, a transcriptome analysis of immune-related genes in parasitized and nonparasitized G. qinghaiensis pupae was performed. A total of 371,260,704 clean reads were assembled into 118,144 unigenes with an average length of 884.33 base pairs. Of these, 23,660 unigenes were annotated in at least one database and 94,484 unigenes were not annotated in any databases. These findings indicated that the majority of the genetic resources (79.97% of all unigenes) in Gynaephora should be further explored. Parasitization significantly affected the transcriptional profile of G. qinghaiensis pupae. The present study identified 12,322 differentially expressed genes and 57 immune-related genes were identified in parasitized G. qinghaiensis pupae. Most immune-related genes were downregulated, potentially resulting from the inhibitory effect of Thektogaster sp. on G. qinghaiensis pupae after parasitization. Overall, the transcriptome analysis sheds valuable light on the molecular mechanisms of G. qinghaiensis parasitization by Thektogaster sp. and promotes the development of novel biocontrol strategies for Gynaephora based on immune defense.

Molecular characterization and expression of six heat shock protein genes in relation to development and temperature in Trichogramma chilonis.

Trichogramma is a kind of egg parasitoid wasp that is widely used to control lepidopterous pests. Temperature is one of the main factors that determines the various life activities of this species, including development, reproduction and parasitism efficiency. Heat shock proteins (HSPs) are highly conserved and ubiquitous proteins that are best known for their responsiveness to temperature and other stresses. To explore the potential role of HSPs in Trichogramma species, we obtained the full-length cDNAs of six HSP genes (Tchsp10, Tchsp21.6, Tchsp60, Tchsp70, Tchsc70-3, and Tchsp90) from T. chilonis and analyzed their expression patterns during development and exposure to temperature stress. The deduced amino acid sequences of these HSP genes contained the typical signatures of their corresponding protein family and showed high homology to their counterparts in other species. The expression levels of Tchsp10, Tchsp21.6 and Tchsp60 decreased during development. However, the expression of Tchsc70-3 increased from the pupal stage to the adult stage. Tchsp70 and Tchsp90 exhibited the highest expression levels in the adult stage. The expression of six Tchsps was dramatically upregulated after 1 h of exposure to 32 and 40°C but did not significantly change after 1 h of exposure to 10 and 17°C. This result indicated that heat stress, rather than cold stress, induced the expression of HSP genes. Furthermore, the expression of these genes was time dependent, and the expression of each gene reached its peak after 1 h of heat exposure (40°C). Tchsp10 and Tchsp70 exhibited a low-intensity cold response after 4 and 8 h of exposure to 10°C, respectively, but the other genes did not respond to cold at any time points. These results suggested that HSPs may play different roles in the development of this organism and in its response to temperature stress.

Profile of Ophiocordyceps sinensis transcriptome and differentially expressed genes in three different mycelia, sclerotium and fruiting body developmental stages.

Ophiocordyceps sinensis, a Chinese complementary and alternative medicine (CAM), is an entomopathogenic, fungus, parasitizing larvae of the moth genus Thitarodes. It has three stages of the life cycle, i.e., the anamorph mycelia prior to infection (Cm_Os), the mycelia sclerotium forming in the caterpillar (Te_Ca), and the fruiting bodies or stromata (Te_St). Characterization of the O. sinensis transcriptome among these stages could provide a better understanding of the underlying biology processes. Transcriptomics of the O. sinensis asexual mycelia and hyphae in deceased caterpillars and perithecial stroma was assessed by using Illumina HiSeq™ 2000 technology. A total of 14,922 unigenes were identified and categorized into 46 sub-categories under three gene ontology categories ("biological process", "cellular component", and "molecular function"). Of these genes, 5520 were differentially expressed among the libraries of these three groups of samples (P < 0.05), and 391 genes occurred in all three groups. Compared to the anamorph stage, there were 3049 differentially expressed genes (DEGs) in the teleomorph stage, but only 1023 DEGs occurred within the teleomorph groups (Te_St vs. Te_Ca). Collectively, this study provides a novel resource to further investigate O. sinensis and their three different development stages.

Transcriptome characterization and gene expression analysis related to chemoreception in Trichogramma chilonis, an egg parasitoid.

Chemoreception is critical for the survival of insects. Insects have a variety of behavioral responses, such as mating, host searching and ovipositing, in response to different odor signals detected in their living environment. Trichogramma chilonis, an egg parasitoid, acts as an efficient and effective biocontrol reagent for many agricultural and forestry insect pests in many parts of China. However, little is known about the molecular mechanism of the olfaction-evoked behavior in T. chilonis. In the present study, we conducted transcriptome profiling analysis of T. chilonis based on the Illumina high-throughput sequencing platform in order to explore differences of chemoreception between male and female T. chilonis. In this study, a total of 85 chemosensory genes were identified from transcriptomic data, including 45 odorant receptors (ORs), 22 odorant binding proteins (OBPs), 14 ionotropic receptors (IRs), 2 sensory neuron membrane proteins (SNMPs) and 2 chemosensory proteins (CSPs). From the analysis of the transcriptome, most of the candidate olfactory genes had similar expression levels in males and females, including a few OR and OBP genes (TchiOR38, TchiOR39, TchiOR40, TchiOR41, TchiOR42, TchiOR43, TchiOR44, TchiOR45, TchiOBP1, TchiOBP4, TchiOBP10, TchiOBP12, TchiOBP18 and TchiOBP19) which showed male-biased expression. Some annotated unigenes were chosen randomly to have qRT-PCR, which verified the correctness of analysis of transcriptome in T. chilonis. This is the first study to obtain and identify candidate genes related to chemoreception in T. chilonis. Our work lays a solid foundation for related future research on the chemosensory system of T. chilonis at the molecular level and helps advance the use of T. chilonis as biological control agents.

Four species of spider genus Cheiracanthium C. L. Koch, 1839 (Araneae, Eutichuridae) from Jinggang Mountains, Jiangxi Province, China.

Four species of spider genus Cheiracanthium C. L. Koch, 1839 are reported from Jinggang Mountains, Jiangxi Province, China. Two of them are described as new to science: C. auriculatumsp. n. (♀♂) and C. echinulatumsp. n. (♂). Cheiracanthium taiwanicum Chen, Huang, Chen & Wang, 2006 is recorded from Mainland China for the first time. Cheiracanthium zhejiangense Hu & Song, 1982, the most similar species to C. auriculatumsp. n., is a newly recorded species of Jiangxi Province. Detailed descriptions, diagnoses, and photographs of the two new species are given. Cheiracanthium taiwanicum and C. zhejiangense are also illustrated.

Molecular Characterization and Expression of Vitellogenin and Vitellogenin Receptor of Thitarodes pui (Lepidoptera: Hepialidae), an Insect on the Tibetan Plateau.

Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in the vitellogenesis of insects. In this study, we cloned and characterized the two corresponding genes (TpVg and TpVgR) in an economically important insect, Thitarodes pui (Lepidoptera: Hepialidae), from the Tibetan plateau. The full length of TpVg is 5566 bp with a 5373 bp open reading frame (ORF) encoding 1,790 amino acids. Sequence alignment revealed that TpVg has three conserved domains: a Vitellogenin_N domain, a DUF1943 domain, and a von Willebrand factor type D domain (VWD). The full length of TpVgR is 5732 bp, with a 5397 bp ORF encoding 1798 amino acids. BLASTP showed that TpVgR belongs to the low-density lipoprotein receptor (LDLR) gene superfamily. Structural analysis revealed that TpVgR has a group of four structural domains: a ligand-binding domain (LBD), an epidermal growth factor (EGF)-precursor homology domain, a transmembrane (TM) domain, and a cytoplasmic domain. In addition, TpVgR has four cysteine-rich LDL repeats in the first ligand-binding site and seven in the second. Quantitative real-time polymerase chain reaction analysis revealed that the expression levels of TpVg and TpVgR are much higher in later pupa than in either the larval or adult stage, implying that the synthesis and uptake of Vg in T. pui occurs in the later pupal stage. These results will help us to understand the molecular mechanism of the reproductive capacity and will provide new insight into the mass rearing and utilization of T. pui.

Cytological Characterization of Anamorphic Fungus Lecanicillium pui and Its Relationship with Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

Ophiocordyceps sinensis (syn. Cordyceps sinensis), one of the most valuable medicinal mushrooms, has great economic importance on the Tibetan Plateau. We isolated an anamorphic fungus Lecanicillium pui from natural O. sinensis specimens and found that the optimal temperature for its culture on potato dextrose agar media was 25°C. Cell ultrastructure in L. pui hyphae and spores was characterized by transmission electron microscopy, and it was observed that some primary organelles showed the typical fungal features. Five chemical elements were determined in this fungus and niobium was discovered for the first time even with trace amounts. A species-specific method, nested polymerase chain reaction, was established to investigate the colonization of this fungus. Thus, the extensive distribution of L. pui on O. sinensis, in the shape of hyphae or mycelia, suggested that it may have subtle and chronic effects on the growth of the O. sinensis teleomorphic stage. These findings provide a potential reference, in the view of microbial ecology, for the study on the occurrence and mechanism of development of O. sinensis.

Transcriptome analysis of Ophiocordyceps sinensis before and after infection of Thitarodes larvae.

Ophiocordyceps sinensis, also referred to as the Chinese caterpillar fungus, is a rare entomopathogenic fungus found in the Qinghai-Tibetan Plateau that is used as a traditional Chinese medicine. O. sinensis parasitizes the larvae of the ghost moth Thitarodes. Characterization of the transcriptome of O. sinensis before and after host infection may provide novel insight into the process by which the fungus interacts with Thitarodes and may help researchers understand how to sustain this valuable resource. In this study, we performed RNA-sequencing (RNA-seq) using Illumina HiSeqTM 2000 technology to generate gene expression profiles of two developmental stages of O. sinensis. Thread-like hyphae before infection and yeast-like hyphal bodies after infection of host larvae were collected for transcriptome analysis. We found that 1640 genes were differentially expressed (q-value < 0.05), of which 818 were upregulated (49.878 %) and 822 were downregulated (50.122 %). Gene ontology (GO) analysis revealed that the differentially expressed genes (DEGs) were especially enriched in terms associated with Biological Process and Molecular Function. Several genes encoding transporter and permease proteins, three glycoside hydrolases, two mycotoxin-related proteins, an antigen protein, and an allergen were identified as being significantly up- or downregulated. Collectively, our findings provide a novel resource for understanding O. sinensis during two critical developmental stages, and offer the opportunity to further investigate the functional mechanisms underlying these stage-specific molecular differences.

Transcriptome characterization and gene expression analysis related to sexual dimorphism in the ghost moth, Thitarodes pui, a host of Ophiocordyceps sinensis.

Thitarodes pui is one of the host species of the Chinese caterpillar fungus Ophiocordyceps sinensis as a traditional Chinese medicine with economic and medical importance. The pupal and adult stages of T. pui are sexually dimorphic. In order to elucidate the molecular mechanisms involved in the sexually dimorphic development of T. pui, we compared the transcriptomes of female and male pupae and adults. We obtained 15,881,734, 16,962,086, 17,514,743, and 17,770,904 clean reads from female pupae, male pupae, female adults, and male adults, respectively. The reads obtained from the four samples were pooled and assembled into 65,165 unigenes, 23,597 of which were annotated. Candidate genes involved in sexual development were identified and analysed. Gene expression analysis revealed that 1406 genes were differentially expressed in male and female pupae, 448 of which were up-regulated in males and 958 were up-regulated in females. A total of 2025 genes were differentially expressed in male and females adults, 1304 of which were up-regulated in males and 721 were up-regulated in females. The functional enrichment of the differentially expressed genes indicated that reproduction and cuticle synthesis were regulated differently between the sexes. The transcriptome data obtained provide significant information regarding the genes involved in sexually dimorphic development, which will improve our understanding of the molecular mechanisms related to sexual dimorphism and helpful for the moth mass rearing which would provide enough host insects for the sustainable utilization of O. sinensis.

Transcriptome response to temperature stress in the wolf spider Pardosa pseudoannulata (Araneae: Lycosidae).

The wolf spider Pardosa pseudoannulata is a dominant predator in paddy ecosystem and an important biological control agent of rice pests. Temperature represents a primary factor influencing its biology and behavior, although the underlying molecular mechanisms remain unknown. To understand the response of P. pseudoannulata to temperature stress, we performed comparative transcriptome analyses of spider adults exposed to 10°C and 40°C for 12 h. We obtained 67,725 assembled unigenes, 21,765 of which were annotated in P. pseudoannulata transcriptome libraries, and identified 905 and 834 genes significantly up- or down-regulated by temperature stress. Functional categorization revealed the differential regulation of transcription, signal transduction, and metabolism processes. Calcium signaling pathway and metabolic pathway involving respiratory chain components played important roles in adapting to low temperature, whereas at high temperature, oxidative phosphorylation and amino acid metabolism were critical. Differentially expressed ribosomal protein genes contributed to temperature stress adaptation, and heat shock genes were significantly up-regulated. This study represents the first report of transcriptome identification related to the Araneae species in response to temperature stress. These results will greatly facilitate our understanding of the physiological and biochemical mechanisms of spiders in response to temperature stress.

Effects of nickel exposure on testicular function, oxidative stress, and male reproductive dysfunction in Spodoptera litura Fabricius.

Nickel is an environmental pollutant that adversely affects the male reproductive system. In the present study, the effects of nickel exposure on Spodoptera litura Fabricius were investigated by feeding larvae artificial diets containing different doses of nickel for three generations. Damage to testes and effects on male reproduction were examined. The amount of nickel that accumulated in the testes of newly emerged males increased as the nickel dose in the diet increased during a single generation. Nickel exposure increased the amount of thiobarbituric acid reactive substances and decreased the amount of glutathione in treatment groups compared with the control. The activity levels of the antioxidant response indices superoxide dismutases, catalase, and glutathione peroxidase in the testes showed variable dose-dependent relationships with nickel doses and duration of exposure. Nickel doses also disrupted the development of the testes by decreasing the weight and volume of testes and the number of eupyrene and apyrene sperm bundles in treatment groups compared with the control. When the nickel-treated males mated with normal females, fecundity was inhibited by the higher nickel doses in all three generations, but fecundity significantly increased during the second generation, which received 5 mg kg(-1) nickel. Hatching rates in all treatments significantly decreased in a dose-dependent manner in the three successive generations. The effects of nickel on these parameters correlated with the duration of nickel exposure. Results indicate assays of testes may be a novel and efficient means of evaluating the effects of heavy metals on phytophagous insects in an agricultural environment.

De novo transcriptome analysis of Thitarodes jiachaensis before and after infection by the caterpillar fungus, Ophiocordyceps sinensis.

Thitarodes jiachaensis is a host species of the pathogenic caterpillar fungus Ophiocordyceps sinensis, which is a fungus with broad medical effects and great economic value. Curated genomic information on Thitarodes is still limited, and the interaction between host Thitarodes larvae and O. sinensis during infection is incompletely understood. In this study, we performed transcriptome sequencing for T. jiachaensis before and after O. sinensis infection using the Illumina sequencing platform, and we identified the transcripts associated with the defense response of T. jiachaensis upon O. sinensis infection. A total of 161,804 transcripts and 94,827 unigenes for T. jiachaensis were obtained from 26.62-Gb clean reads, and 35.03% of all the unigenes were annotated in current databases. The expression of 1581 genes was significantly altered following infection; among them, 928 (58.70%) were up-regulated and 653 (41.30%) were down-regulated. Genes encoding physical barriers such as cuticle proteins and peritrophic matrix proteins, antimicrobial peptides (AMPs), pattern recognition receptors (PRRs), and enzymes in the proteolytic cascade were predicted to be involved in the response of T. jiachaensis to O. sinensis infection. Together, these data provide a valuable genomic resource for further studies of Thitarodes and increase our understanding of the host-pathogen interaction that occurs between Thitarodes and O. sinensis.

SEASONAL CHANGES OF FATTY ACID COMPOSITION IN Thitarodes pui Larvae, A HOST OF Ophiocordyceps sinensis.

BACKGROUND: Thitarodes larvae are the host of Ophiocordyceps sinensis and exist in the permafrost region of the Tibetan Plateau. OBJECTIVE: To understand the adaptation mechanism of Thitarodes larvae to seasonal fluctuations of ambient temperatures in the Tibetan Plateau by studying seasonal changes of the fatty acids composition in the larvae of T. pui. MATERIALS AND METHODS: The profile of fatty acids in the 6th instar T. pui larvae collected at the mid-month in a whole year were examined by GC-MS. RESULTS: There was a negative correlation between the total lipid and ambient (soil) temperature. Further study indicated that oleic, palmitic, linoliec, palmitoleic, stearic were the major fatty acids. The ratio of unsaturated fatty acids to saturated fatty acids (U/S) and the unsaturated index (UI) in triacylglycerols remain stable during the whole year, while the U/S and UI in phospholipids vary dramatically in response to soil temperature. CONCLUSION: The fluctuations in phospholipids were attributed to seasonal changes of oleic and linoleic. The changes of the fatty acid composition may result from their adaptation to the variation of temperature in different seasons.

Development of Ophiocordyceps sinensis through Plant-Mediated Interkingdom Host Colonization.

Ophiocordyceps sinensis is a well-known entomogenous and medicinal fungus. After its anamorphs parasitize the larvae of the genus Thitarodes, fruit-bodies may form to be used as medicine. However, its developmental mechanisms remain unknown. The distribution of O. sinensis was determined in different tissues of the Thitarodes larvae and the dominant plant species using real-time quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) technique, respectively. We found that more fungal material was located in plants than in larvae, especially in Ranunculus tanguticus. A considerable amount was detected in larval intestinal-wall and plant roots. It is suggested that plants are the potential hosts of O. sinensis, which modifies our understanding of the life cycle of O. sinensis and indicates that the phytophagous larvae may become infected as they feed. Our research may contribute to the study of systematic evolution and population ecology of O. sinensis, elucidate its developmental mechanism and promote sustainable harvesting.

Metabolic characterization of natural and cultured Ophicordyceps sinensis from different origins by 1H NMR spectroscopy.

Ophicordyceps sinensis is a well-known traditional Chinese medicine and cultured mycelium is a substitute for wild O. sinensis. Metabolic profiles of wild O. sinensis from three geographical locations and cultivated mycelia derived from three origins were investigated using (1)H nuclear magnetic resonance (NMR) analysis combined with multivariate statistical analysis. A total of 56 primary metabolites were identified and quantified from O. sinensis samples. The principle component analysis (PCA) showed significant differences between natural O. sinensis and fermentation mycelia. Seven metabolites responsible for differentiation were screened out by orthogonal partial least squares discriminant analysis (OPLS-DA). The concentrations of mannitol, trehalose, arginine, trans-4-hydroxyproline, alanine and glucitol were significantly different between wild and cultured groups. The variation in metabolic profiling among artificial mycelia was greater than that among wild O. sinensis. Furthermore, wild samples from different origins were clearly distinguished by the levels of mannitol, trehalose and some amino acids. This study indicates that (1)H NMR-based metabolomics is useful for fingerprinting and discriminating O. sinensis of different geographical regions and cultivated mycelia of different strains. The present study provided an efficient approach for investigating chemical compositions and evaluating the quality of medicine and health food derived from O. sinensis.